Renzhi Cai

Inhibition of growth of MDA-MB-231 human breast cancer xenografts in nude mice by bombesin/gastrin-releasing peptide (GRP) antagonists RC-3940-II and RC-3095

Bombesin or gastrin-releasing peptide (GRP) may act as autocrine growth factors and play a role in the initiation and progression of breast cancer. We investigated the effect of bombesin/GRP antagonists RC-3095 and RC-3940-II on the growth of the MDA-MB-231 oestrogen-independent human breast cancer cell line xenografted into female nude mice. Bombesin/GRP antagonists, RC-3095 and RC-3940-II, were administered subcutaneously twice daily at a dose of 10 micrograms for 5 weeks. The growth of MDA-MB-231 tumours was inhibited during the treatment, as shown by a reduction in tumour volume. RC-3940-II and RC-3095 significantly decreased the final tumour volume by 72.4% and 57.7%, respectively, and greatly reduced tumour weights. RC-3940-II also significantly increased tumour doubling time and appeared to be more effective than RC-3095 in inhibiting the growth of MDA-MB-231 breast cancers. Serum gastrin and insulin-like growth factor-I (IGF-I) levels in animals treated with RC-3095 or RC-3940-II showed no significant changes as compared with controls. There was a significant decrease in the number of binding sites for epidermal growth factor (EGF), as well as bombesin, in tumour cells after chronic treatment with RC-3095 or RC-3940-II, which might be related to inhibition of tumour growth. Reverse transcription polymerase chain reaction, followed by Southern blot analysis, also showed a reduction in the expression of mRNA for EGF receptors in the group treated with RC-3940-II. Our findings suggest that bombesin/GRP antagonists such as RC-3095 or RC-3940-II could be considered for endocrine therapy for oestrogen-independent breast cancers, but further investigations are necessary.

Effects of somatostatin analogue RC-160 and bombesin/gastrin-releasing peptide antagonists on the growth of human small-cell and non-small-cell lung carcinomas in nude mice

We investigated the effects of our synthetic bombesin/gastrin-releasing peptide (GRP) antagonists and somatostatin analogue RC-160 on the growth of human small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (non-SCLC) lines in nude mice. Athymic nude mice bearing xenografts of the SCLC NCl-H69 line or non-SCLC NCl-H157 line were treated for 5 and 4 weeks, respectively, with somatostatin analogue RC-160 or various bombesin/GRP antagonists. RC-160, administered s.c. peritumorally at a dose of 100 micrograms per animal per day, inhibited the growth of H69 SCLC xenografts as shown by more than 70% reduction in tumour volumes and weights, as compared with the control group. Bombesin/GRP antagonists, RC-3440, RC-3095 and RC-3950-II, given s.c. peritumorally at a dose of 20 micrograms per animal per day, also inhibited the growth of H69 SCLC tumours. RC-3950-II had the greatest inhibitory effect and decreased tumour volume and weights by more than 80%. The growth of H-157 non-SCLC xenografts was significantly reduced by treatment with RC-160, but not with bombesin/GRP antagonist RC-3095. In mice bearing either tumour model, administration of RC-160 significantly decreased serum growth hormone and gastrin levels. Specific high-affinity receptors for bombesin and somatostatin were found on membranes of SCLC H69 tumours, but not on non-SCLC H157 tumours. Receptor analyses demonstrated high-affinity binding sites for epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the membranes of H69 and H157 tumours. EGF receptors were down-regulated on H69 tumours after treatment with RC-160 and bombesin/GRP antagonists. The concentration of binding sites for EGF and IGF-I on the H157 tumours was decreased after treatment with RC-160, but bombesin/GRP antagonist RC-3095 had no effect. These results demonstrate that bombesin/GRP antagonists inhibit the growth of H-69 SCLC, but not of H-157 non-SCLC xenografts in nude mice, whereas somatostatin analogue RC-160 is effective in both tumour models. This raises the possibility that these peptide analogues could be used selectively in the treatment of various subclasses of lung cancer.

Inhibition of growth of MDA-MB-468 estrogen-independent human breast carcinoma by bombesin/gastrin-releasing peptide antagonists RC-3095 and RC-3940-II.

The growth of breast carcinoma is promoted by autocrine growth factors such as the bombesin (BN)‐like peptides and epidermal growth factor (EGF). The stimulatory action of BN‐like peptides can be blocked by the use of BN/gastrin‐releasing peptide (GRP) antagonists.

Inhibitory effect of bombesin/gastrin-releasing peptide (GRP) antagonists RC-3950-II and RC-3095 on MCF-7 MIII human breast cancer xenografts in nude mice.

Bombesin/gastrin-releasing peptide (GRP) may be involved in the growth of human breast cancers. Nude mice bearing xenografts of MCF-7 MIII human breast cancer cell line were treated for 7 weeks with bombesin/GRP antagonists RC-3950-II and RC-3095. RC-3950-II, administered sc twice daily at a dose of 10 micrograms, produced significant inhibitory effects on tumor growth after 2 weeks of administration. RC-3095 acetate (D 22213), injected sc twice daily at the same dose of 10 micrograms, suppressed tumor growth after 4 weeks. Both RC-3950-II and RC-3095 significantly decreased the final tumor volume and tumor weights. RC-3950-II appeared to be somewhat more efficacious than RC-3095 in inhibiting the growth of MCF-7 MIII breast cancers. Chronic treatment with either bombesin/GRP antagonist caused down-regulation of receptors for epidermal growth factor (EGF) in tumor cell membranes, which might be related to inhibition of tumor growth. These findings suggest that bombesin/GRP antagonists should be considered for a new endocrine therapy of breast cancer.

Endocrine, Gastrointestinal, and Antitumor Activity of Somatostatin Analogues

Since recent studies with somatostatin were carefully reviewed by Reichlin (1983) and by a variety of authors in the 2nd International Symposium on Somatostatin, which was held in Athens in 1981 but published with updates in 1984 (Raptis et al., 1984), no attempts are made to cover new developments with somatostatin-14 and somatostatin-28. Instead, this chapter focuses on the recent work in the field of somatostatin analogues and the investigations of their endocrine, gastrointestinal, and antitumor activities.

Activation of mitogen-activated protein kinases by a splice variant of GHRH receptor

Hypothalamic GHRH controls the release of GH from the pituitary gland and also acts as a growth factor in a variety of cancers. The mitogenetic activity of GHRH is exerted through the binding to the pituitary type receptor (pGHRH-R) and its splice variants, mainly SV1. The intracellular signaling pathways that are activated upon the binding of GHRH to the SV1 receptor have not been elucidated. HeLa cervical cancer cells do not express GHRH or GHRH receptors (GHRHRs) and thus do not respond to GHRH or GHRH antagonists. In order to elucidate the mechanism of action of SV1 receptor, we transfected HeLa cells with plasmids for pcDNA3-GHRHR or pcDNA3-SV1. The transfected cells responded to both GHRH (1-29)NH(2) and GHRH antagonist MZ-5-156, as shown by an increase or decrease respectively in the proliferation rate in vitro and the expression of proliferative cell nuclear antigen. We also demonstrated that when the cells transfected with SV1 plasmid are stimulated with GHRH (1-29)NH(2), SV1 receptor activates the mitogen-activated protein kinases pathway (MAPKs), as shown previously for the cells that express pGHRH-R. Our results show, for the first time, the activation of the MAPKs cascade by the SV1 receptor. Since SV1 receptor is found in various tumors and mediates the responses to GHRH and synthetic antagonists, our findings shed light on the mechanism of action of SV1 receptor in cancer cells.

Growth hormone-releasing hormone receptor antagonists inhibit human gastric cancer through downregulation of PAK1–STAT3/NF-κB signaling

Significance Gastric cancer (GC) is a leading and lethal malignancy in the world. Unfortunately, therapeutics targeting GC lag behind those of many other cancers, and the effective treatment options are limited. Here we report that increased expression of growth hormone-releasing hormone receptor (GHRH-R) in tumor specimens is significantly associated with tumorigenic progression and poor clinical outcome of GC patients. MIA-602, a highly potent GHRH-R antagonist, effectively inhibits GC growth in vitro and in vivo. The anti-neoplastic effects were mediated by blockage of the p21-activated kinase 1 (PAK1)–signal transducer and activator of transcription 3 (STAT3)/nuclear factor-κB (NF-κB) axis. Thus, GHRH-R presents a molecular marker and therapeutic target of GC, and MIA-602 demonstrates excellent therapeutic potential against human GC. Gastric cancer (GC) ranks as the fourth most frequent in incidence and second in mortality among all cancers worldwide. The development of effective treatment approaches is an urgent requirement. Growth hormone-releasing hormone (GHRH) and GHRH receptor (GHRH-R) have been found to be present in a variety of tumoral tissues and cell lines. Therefore the inhibition of GHRH-R was proposed as a promising approach for the treatment of these cancers. However, little is known about GHRH-R and the relevant therapy in human GC. By survival analyses of multiple cohorts of GC patients, we identified that increased GHRH-R in tumor specimens correlates with poor survival and is an independent predictor of patient prognosis. We next showed that MIA-602, a highly potent GHRH-R antagonist, effectively inhibited GC growth in cultured cells. Further, this inhibitory effect was verified in multiple models of human GC cell lines xenografted into nude mice. Mechanistically, GHRH-R antagonists target GHRH-R and down-regulate the p21-activated kinase 1 (PAK1)-mediated signal transducer and activator of transcription 3 (STAT3)/nuclear factor-κB (NF-κB) inflammatory pathway. Overall, our studies establish GHRH-R as a potential molecular target in human GC and suggest treatment with GHRH-R antagonist as a promising therapeutic intervention for this cancer.

Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocin-induced NOD/SCID mice

Significance Improved therapeutic strategies for transplantation of pancreatic islet cells to selected patients with type-1 diabetes are urgently needed. Growth hormone-releasing hormone (GHRH) agonists to influence growth, function, and engraftment of islet cells were previously reported by us. This study demonstrates greater stimulation by improved GHRH agonists, on proliferation, gene expressions, and the signaling pathways in pancreatic β cells. Agonist MR-409 in vivo reduced the severity of streptozotocin-induced diabetes in nonobese diabetic severe combined immunodeficiency mice. Transplantation of rat islets preconditioned in vitro with MR-409 and its administration in vivo promoted growth, function, and engraftment of exogenous islets, supporting the use of GHRH agonists in type-1 diabetes. The beneficial effects of GHRH agonists on the functions of β cells may also provide approaches to their application in type-2 diabetes. Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote growth, function, and engraftment of islet cells following transplantation. Here we evaluated recently synthesized GHRH agonists on the proliferation and biological functions of rat pancreatic β-cell line (INS-1) and islets. In vitro treatment of INS-1 cells with GHRH agonists increased cell proliferation, the expression of cellular insulin, insulin-like growth factor-1 (IGF1), and GHRH receptor, and also stimulated insulin secretion in response to glucose challenge. Exposure of INS-1 cells to GHRH agonists, MR-356 and MR-409, induced activation of ERK and AKT pathways. Agonist MR-409 also significantly increased the levels of cellular cAMP and the phosphorylation of cAMP response element binding protein (CREB) in INS-1 cells. Treatment of rat islets with agonist, MR-409 significantly increased cell proliferation, islet size, and the expression of insulin. In vivo daily s.c. administration of 10 μg MR-409 for 3 wk dramatically reduced the severity of streptozotocin (STZ)-induced diabetes in nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice. The maximal therapeutic benefits with respect to the efficiency of engraftment, ability to reach normoglycemia, gain in body weight, response to high glucose challenge, and induction of higher levels of serum insulin and IGF1 were observed when diabetic mice were transplanted with rat islets preconditioned with GHRH agonist, MR-409, and received additional treatment with MR-409 posttransplantation. This study provides an improved approach to the therapeutic use of GHRH agonists in the treatment of diabetes mellitus.

Synthesis and structure-activity studies on novel analogs of human growth hormone releasing hormone (GHRH) with enhanced inhibitory activities on tumor growth

HighlightsWe report synthesis of new GHRH analogs with increased anticancer activity.Incorporation of pentafluoro Phe and Orn12,21 into GHRH analogs augments antitumor activity.ω‐amino acids at N‐and C‐terminus produce an increased inhibition of tumor growth.GHRH analogs inhibit growth of prostatic and other cancers in vitro and in vivo.GHRH analogs target G protein‐coupled GHRH receptors. Abstract The syntheses and biological evaluations of new GHRH analogs of Miami (MIA) series with greatly increased anticancer activity are described. In the design and synthesis of these analogs, the following previous substitutions were conserved: D‐Arg2, Har9, Abu15, and Nle27. Most new analogs had Ala at position 8. Since replacements of both Lys12 and Lys21 with Orn increased resistance against enzymatic degradation, these modifications were kept. The substitutions of Arg at both positions 11 and 20 by His were also conserved. We kept D‐Arg28, Har29 –NH2 at the C‐terminus or inserted Agm or 12‐amino dodecanoic acid amide at position 30. We incorporated pentafluoro‐Phe (Fpa5), instead of Cpa, at position 6 and Tyr(Me) at position 10 and ω‐amino acids at N‐terminus of some analogs. These GHRH analogs were prepared by solid‐phase methodology and purified by HPLC. The evaluation of the activity of the analogs on GH release was carried out in vitro on rat pituitaries and in vivo in male rats. Receptor binding affinities were measured in vitro by the competitive binding analysis. The inhibitory activity of the analogs on tumor proliferation in vitro was tested in several human cancer cell lines such as HEC‐1A endometrial adenocarcinoma, HCT‐15 colorectal adenocarcinoma, and LNCaP prostatic carcinoma. For in vivo tests, various cell lines including PC‐3 prostate cancer, HEC‐1A endometrial adenocarcinoma, HT diffuse mixed &bgr; cell lymphoma, and ACHN renal cell carcinoma cell lines were xenografted into nude mice and treated subcutaneously with GHRH antagonists at doses of 1–5 &mgr;g/day. Analogs MIA‐602, MIA‐604, MIA‐610, and MIA‐640 showed the highest binding affinities, 30, 58, 48, and 73 times higher respectively, than GHRH (1‐29) NH2. Treatment of LNCaP and HCT‐15 cells with 5 &mgr;M MIA‐602 or MIA‐690 decreased proliferation by 40%–80%. In accord with previous tests in various human cancer lines, analog MIA‐602 showed high inhibitory activity in vivo on growth of PC‐3 prostate cancer, HT‐mixed &bgr; cell lymphoma, HEC‐1A endometrial adenocarcinoma and ACHN renal cell carcinoma. Thus, GHRH analogs of the Miami series powerfully suppress tumor growth, but have only a weak endocrine GH inhibitory activity. The suppression of tumor growth could be induced in part by the downregulation of GHRH receptors levels.

Agonistic analogs of growth hormone releasing hormone (GHRH) promote wound healing by stimulating the proliferation and survival of human dermal fibroblasts through ERK and AKT pathways

Decreased or impaired proliferation capability of dermal fibroblasts interferes with successful wound healing. Several growth factors tested failed to fully restore the growth of fibroblasts, possibly due to their rapid degradation by proteases. It is therefore critical to find new agents which have stimulatory effects on fibroblasts while being highly resistant to degradation. In such a scenario, the activities of two agonistic analogs of growth hormone releasing hormone (GHRH), MR-409 and MR-502, were evaluated for their impact on proliferation and survival of primary human dermal fibroblasts. In vitro, both analogs significantly stimulated cell growth by more than 50%. Under serum-depletion induced stress, fibroblasts treated with MR-409 or MR-502 demonstrated better survival rates than control. These effects can be inhibited by either PD98059 or wortmannin. Signaling through MEK/ERK1/2 and PI3K/AKT in an IGF-1 receptor-independent manner is required. In vivo, MR-409 promoted wound closure. Animals treated topically with MR-409 healed earlier than controls in a dose-dependent manner. Histologic examination revealed better wound contraction and less fibrosis in treated groups. In conclusion, MR-409 is a potent mitogenic and anti-apoptotic factor for primary human dermal fibroblasts. Its beneficial effects on wound healing make it a promising agent for future development.